Stabilized tetrazolium-phenazine reagent compositions and methods for using the same

ABSTRACT

Stabilized tetrazolium dye-phenazine reagent compositions and methods for their use in the measurement of an analyte in a sample are provided. The subject reagent compositions include: (1) a tetrazolium dye component, e.g., a water soluble tetrazolium salt; (2) a phenazine component; and (3) an effective amount of one or more tetrazolium dye-phenazine stabilizing reagents, e.g., an inorganic Group IIIA compound and/or a flavin. In many embodiments, the subject reagent compositions include additional members of an analyte oxidizing signal producing system, such as: an analyte oxidizing enzyme, e.g., an analyte dehydrogenase or an analyte oxidase; and an enzyme cofactor. Also provided are test strips that include the subject reagent compositions, as well as systems and kits incorporating the subject test strips. The subject reagent compositions, test strips, systems and kits find use in the detection of a wide variety of analytes in a sample, such as a physiological sample, e.g., blood or a fraction thereof, or ISF (interstitial fluid).

INTRODUCTION

[0001] 1. Field of the Invention

[0002] The field of this invention is analyte measurement

[0003] 2. Background of the Invention

[0004] Analyte measurement in physiological fluids, e.g., blood orblood-derived products, is of ever increasing importance to today'ssociety. Analyte detection assays find use in a variety of applications,including clinical laboratory testing, home testing, etc., where theresults of such testing play a prominent role in diagnosis andmanagement in a variety of disease conditions. Analytes of interestinclude alcohol, formaldehyde, glucose, glutamic acid, glycerol,beta-hydroxybutyrate, L-lactate, leucine, malic acid, pyruvic acid,steroids, etc. In response to this growing importance of analytemeasurement, a variety of analyte measurement protocols and devices forboth clinical and home use have been developed.

[0005] Many of the protocols and devices that have been developed todate employ a signal producing system to identify the presence of theanalyte of interest in a physiological sample, such as blood.

[0006] While a variety of such signal producing systems have beendeveloped to date for use in the measurement of a wide variety ofdifferent analytes, there continues to be a need for the furtherdevelopment of such systems.

[0007] Relevant Literature

[0008] Patent documents of interest include: U.S. Pat. No. 6,200,773;U.S. Pat. No. 5,902,731; U.S. Pat. No. 4,613,569, U.S. Pat. No.4,847,196; EP 0 908 453 A1; WO 94/01578 and WO 94/01544.

SUMMARY OF THE INVENTION

[0009] Stabilized tetrazolium dye-phenazine reagent compositions thatinclude a tetrazolium dye reagent, e.g., a water soluble tetrazoliumsalt, and an effective amount of one or more tetrazolium dye-phenazinestabilizing reagents, e.g., an inorganic Group IIIA compound and/or aflavin, are provided. In many embodiments, the reagent compositionsinclude members of analyte oxidizing signal producing system of whichthe tetrazolium dye and phenazine reagents are members, which systemincludes one or more of the following additional components: an analyteoxidizing enzyme, e.g., an analyte dehydrogenase or an analyte oxidase;and an enzyme cofactor. Also provided are test strips that include thesubject reagent compositions, as well as systems and kits incorporatingthe subject test strips. The subject reagent compositions, test strips,systems and kits find use in the measurement of a wide variety ofanalytes in a sample, such as a physiological sample, e.g., blood or afraction thereof, or ISF (interstitial fluid).

BRIEF DESCRIPTION OF THE FIGURES

[0010]FIG. 1 provides characterization results of a test strip having areagent composition with and without borax.

[0011]FIG. 2 provides characterization results of a test strip having areagent composition with and without FAD.

[0012]FIG. 3 provides characterization results of a test strip having areagent composition with and without borax and FAD.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0013] Stabilized tetrazolium dye-phenazine reagent compositions andmethods for their use in the measurement of an analyte in a sample areprovided. The subject reagent compositions include: (1) a tetrazoliumdye component, e.g., a water soluble tetrazolium salt; (2) a phenazinecomponent; and (3) an effective amount of one or more tetrazoliumdye-phenazine stabilizing reagents, e.g., an inorganic Group IIIAcompound and/or a flavin. In many embodiments, the subject reagentcompositions include additional members of an analyte oxidizing signalproducing system, such as: an analyte-oxidizing enzyme, e.g., an analytedehydrogenase or an analyte oxidase; and an enzyme cofactor. Alsoprovided are test strips that include the subject reagent compositions,as well as systems and kits incorporating the subject test strips. Thesubject reagent compositions, test strips, systems and kits find use inthe detection of a wide variety of analytes in a sample, such as aphysiological sample, e.g., blood or a fraction thereof, or ISF(interstitial fluid).

[0014] Before the subject invention is described further, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

[0015] In this specification and the appended claims, the singular forms“a,” “an” and “the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

[0016] Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range, and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

[0017] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this invention belongs. Although any methods,devices and materials similar or equivalent to those described hereincan be used in the practice or testing of the invention, the preferredmethods, devices and materials are now described.

[0018] All publications mentioned herein are incorporated herein byreference for the purpose of describing and disclosing the cell lines,vectors, and methodologies, which are described in the publications,which might be used in connection with the presently describedinvention.

[0019] As summarized above, the subject invention provides stabilizedtetrazolium dye compositions and methods for their use, as well asreagent test strips, systems and kits. In further describing theinvention, each of these inventive features is discussed in greaterdetail below.

[0020] Reagent Compositions

[0021] As summarized above, the subject invention provides stabilizedtetrazolium dye-phenazine reagent compositions, which compositions finduse in detecting a wide variety of analytes in a sample. The subjecttetrazolium dye-phenazine reagent compositions of the present inventionare characterized by at least including a tetrazolium dye reagent; aphenazine electron transfer reagent and an effective amount of one ormore tetrazolium dye-phenazine stabilizing reagents, e.g., an inorganicGroup IIIA compound and/or a flavin.

[0022] The tetrazolium dye reagent is a tetrazolium compound (dyeprecursor) that, upon acceptance of a transferred hydride, forms acolored formazan product. In many embodiments, the tetrazolium dyereagent is a water soluble tetrazolium salt that is capable of acceptinga hydride to produce a water soluble, colored formazan product. Watersoluble tetrazolium salts of interest include those described in EP 0908 453, the disclosure of which is herein incorporated by reference.One class of water soluble tetrazolium salts of interest include thosedescribed by formula 2 on page 2, lines 35 to 48 of EP 0 908 453.Another class of water soluble tetrazolium salts of interest includethose described by formula 1 on page 3, lines 10-25 of EP 0 908 453.

[0023] Specific water soluble tetrazolium compounds or salts that are ofparticular interest include, but are not limited to:2-(2′benzothiazolyl)-5-styryl-3-(4′-phthalhydrazidyl) tetrazolium(BSPT), 2-benzothiazolyl-(2)-3,5-diphenyl tetrazolium (BTDP),2,3-di(4-nitrophenyl) tetrazolium (DNP), 2,5-diphenyl-3-(4-styrylphenyl)tetrazolium (DPSP), distyryl nitroblue tetrazolium (DS-NBT),3,3′-[3,3′-dimethoxy-(1,1′-biphenyl)-4,4′-diyl]-bis[2-(4-nitrophenyl)-5-phenyl(-2Htetrazolium (NBT), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazolium (MTT), 2-phenyl-3-(4-carboxyphenyl)-5-methyl tetrazolium(PCPM), tetrazolium blue (TB), thiocarbamyl nitroblue tetrazolium(TCNBT), tetranitroblue tetrazolium (TNBT), tetrazolium violet, (TV),2-benzothiazothiazolyl-3-(4-carboxy-2-methoxyphenyl)-5-[4-(2-sulfoethylcarbamoyl)phenyl]-2H-tetrazolium (WST-4), and2,2′-dibenzothiazolyl-5,5′-bis[4-di(2-sulfoethyl)carbamoylphenyl]-3,3′-(3,3′-dimethoxy-4,4′-biphenylene)ditetrazolium, disodium salt (WST-5). Incertain embodiments, the dye compounds is selected from the group of:2,2′-dibenzothiazolyl-5,5′-bis[4-di(2-sulfoethyl)carbamoylphenyl]-3,3-(3,3-dimethoxy-4,4′-biphenylene)ditetrazolium,disodium salt (WST-5);2-benzothiazolyl-3-(4-carboxy-2-methoxyphenyl)-5-[4-(2-sulfoethylcarbamoyl)phenyl]-2H-tetrazolium(WST-4) and the like. WST-5 is preferred in many embodiments because itreadily dissolves in an aqueous medium, which is most compatible withbiological samples. Furthermore, the resulting formazan compoundexhibits strong spectral absorption at the purple-blue region, thusreducing the need for correcting the background signal from hemoglobin.While the amount of the tetrazolium dye reagent may vary depending onthe nature of the reagent composition, e.g., whether it is in dry or wetform, the concentration of the dye reagent in many embodiments rangesfrom about 1.5 mM to about 50 mM, usually from about 3 mM to about 40 mMand more usually from about 3.5 mM to about 28 mM.

[0024] The subject reagent compositions also include a phenazineelectron transfer agent. By phenazine electron transfer agent is meant aphenazine compound or molecule that can transfer an electron, in theform of a hydride ion, from a reduced enzyme cofactor to the watersoluble tetrazolium product. Specific phenazine compounds of interestinclude, but are not limited to: phenazine, phenazine methosulfate(PMS), phenazine ethosulfate, methoxyphenazine methosulfate andsafranine. While the amount of the phenazine electron transfer reagentmay vary depending on the nature of the reagent composition, e.g.,whether it is in dry or wet form, the concentration of the phenazinereagent in many embodiments ranges from about 0.01 mM to about 50 mM,usually from about 0.05 mM to about 10 mM and more usually from about0.1 mM to about 5 mM.

[0025] In addition to the tetrazolium dye and phenazine componentsdescribed above, the subject reagent compositions also include aneffective amount of one or more tetrazolium dye-phenazine stabilizingreagents, e.g., an inorganic Group IIIA compound and/or a flavin. Assuch, in certain embodiments, the subject reagent compositions includean effective amount of a Group IIIA compound. In other embodiments, thesubject compositions include an effective amount of a flavin. In yetother embodiments, the subject compositions include effective amounts ofboth a Group IIIA compound and a flavin. By effective amount is meant anamount sufficient to stabilize the tetrazolium-phenazine system so thatit provides a substantially greater optical density signal after atleast about 10 sec than a system that does not include the stabilizingagent(s), as measured using the evaluation protocol reported in theExperimental Section, below. An optical density is considered to besubstantially greater than a control (where no stabilizing agent isemployed) if, at the 10 sec point, it is at least about 2 fold, usuallyabout 2.14 fold greater than the control.

[0026] Any convenient Group IIIA compound may be employed which providesfor the desired stabilization. Representative Group III compounds arethose include elements of the Group IIIA column of the periodic table,e.g., B, Al, Ga, In, Tl, where in many embodiments, the Group IIIAcompound is a boron or aluminum compound, where of particular interestare inorganic compounds that include these elements, e.g., borates (orboric acid), aluminates, etc., where in many embodiments the compound isa borate, e.g., borax, or boric acid. The ratio of Group IIIAstabilizing component to tetrazolium dye in the composition typicallyranges from about 50 to about 800, usually from about 100 to about 400.As such, in many embodiments, the concentration of the Group IIIAstabilizing reagent in the composition ranges from about 0.1 M to about1.2 M, usually from about 0.2 M to about 1 M.

[0027] Any convenient flavin compound may be employed which provides forthe desired stabilization. Representative flavin compounds are FMN andFAD. The ratio of flavin stabilizing component to tetrazolium dye in thecomposition typically ranges from about 0.02 to about 17, usually fromabout 0.03 to about 1. As such, in many embodiments, the concentrationof the flavin stabilizing reagent in the composition ranges from about 1mM to about 25 mM, usually from about 2 mM to about 15 mM.

[0028] Where both a Group IIIA and flavin stabilizing agent are presentin the composition, the ratio of the amounts of these two agentstypically ranges from about 2 to about 800, usually from about 10 toabout 400.

[0029] The presence of the stabilizing agent(s) described abovestabilizes the tetrazolium dye-phenazine system, as described above,with respect to heat (56° C.) exposure of at least about one week.

[0030] As mentioned above, the subject reagent compositions typicallyfurther include additional members of an analyte oxidizing signalproducing system. By signal producing system is meant a collection oftwo or more compounds or molecules which are capable of acting inconcert, when combined, to produce a detectable signal that isindicative of the presence of, and often amount of, a particular analytein a given sample. The term signal producing system is used broadly toencompass both a mixture of all of the reagent constituents of thesignal producing system as well as a system in which one or more of thereagent constituents are separated from the remainder of the reagentconstituents, e.g., as is present in a kit.

[0031] As mentioned above, the signal producing system of the subjectcompositions is an analyte oxidizing signal producing system. Theanalyte oxidizing agent is generally an enzyme that is capable ofremoving a hydride from the analyte of interest to produce an oxidizedform of the analyte. Analyte oxidizing enzymes of interest includesanalyte oxidases and analyte dehydrogenases. Analyte oxidases ofinterest include, but are not limited to: glucose oxidase (where theanalyte is glucose); cholesterol oxidase (where the analyte ischolesterol); alcohol oxidase (where the analyte is alcohol); bilirubinoxidase (where the analyte is bilirubin); choline oxidase (where theanalyte is choline); formaldehyde dehydrogenase (where the analyte isformaldehyde); glutamate oxidase (where the analyte is L-glutamic acid);glycerol oxidase (where the analyte is glycerol); galactose oxidase(where the analyte is galactose); L-ascorbate oxidase (where the analyteis ascorbic acid); lactate oxidase (where the analyte is lactic acid);leucine oxidase (where the analyte is leucine); malate oxidase (wherethe analyte is malic acid); pyruvate oxidase (where the analyte ispyruvic acid); urate oxidase (where the analyte is uric acid); and thelike.

[0032] Analyte dehydrogenases of interest include, but are not limitedto: alcohol dehydrogenase for alcohol; formaldehyde dehydrogenase forformaldehyde; glucose dehydrogenase for glucose; glucose-6-phosphatedehydrogenase for glucose-6-phosphate; glutamate dehydrogenase forglutamic acid; glycerol dehydrogenase for glycerol; beta-hydroxybutyratedehydrogenase for beta-hydroxybutyrate; hydroxysteroid dehydrogenase forsteroid; L-lactate dehydrogenase for L-lactate; leucine dehydrogenasefor leucine; malate dehydrogenase for malic acid, and pyruvatedehydrogenase for pyruvic acid.

[0033] In many embodiments, the subject signal producing systems alsoinclude an enzyme cofactor that is capable of interacting with theoxidizing agent in a manner such that the analyte of interest isoxidized by the oxidizing agent, which agent concomitantly reduces theenzyme cofactor. Enzyme cofactors of interest include, but are notlimited to: i.e. beta-nicotinamide adenine dinucleotide (beta-NAD);beta-nicotinamide adenine dinucleotide phosphate (beta-NADP);thionicotinamide adenine dinucleotide; thionicotinamide adeninedinucleotide phosphate; nicotinamide 1,N6-ethenoadenine dinucleotide;nicotinamide 1,N6-ethenoadenine dinucleotide phosphate; andpyrrolo-quinoline quinone (PQQ); and flavin compounds, FAD, FMN etc.Enzyme cofactors of particular interest that may be included in thesubject signal producing systems include: PQQH₂, NADH or NAD(P)H.

[0034] As indicated above, the subject compositions are present aseither wet or dry compositions. By wet composition is meant a fluidcomposition, typically an aqueous composition. Such compositions finduse in various assay configurations, such as cuvette configurations,which are well known in the art. By dry compositions is meant acomposition that is not fluid, i.e., in dry form, such as a compositionthat is substantially free of uncombined water. Such compositions aretypically found in reagent test strips, as described in greater detailbelow.

[0035] Reagent Test Strips

[0036] Of particular interest in many embodiments of the subjectinvention are reagent test strips that include the above described dryreagent compositions and are intended for use in measuring the presenceor concentration of an analyte in a sample. In particular, the inventionprovides dry strips for assaying for a particular analyte in wholeblood, e.g., beta-hydroxybutyrate, glucose, etc. In the broadest sense,the reagent test strip includes a solid support and a dry reagentcomposition present thereon, where the dry reagent composition is madeup of all of the reagent compounds necessary to produce a detectablesignal in the presence of the analyte of interest. In most embodimentsof the subject invention, the dry reagent composition present on thesubject test strip is one that includes the following members: ananalyte oxidizing enzyme, an enzyme cofactor, an electron transferagent, a water soluble tetrazolium salt, and borate (or boric acid)and/or flavinstabilizing reagents, where each of these constituentmembers are described in greater detail above.

[0037] In many embodiments, the subject test strips include a membranetest pad that is affixed to a solid support. The support may be aplastic—e.g., polystyrene, nylon, or polyester—or metallic sheet or anyother suitable material known in the art. Associated with the test pad,e.g., coated onto the test pad, incorporated into the test pad, etc., isthe reagent composition. The strip may also be configured in morecomplex arrangements, e.g., where the test pad is present between thesupport and a surface layer, where one or more reagents employed insample processing may be present on the surface layer. In addition, flowpaths or channels may be present on the test strip, as is known in theart. Of interest in many embodiments is the test strip configurationsdisclosed in U.S. Pat. No. 5,902,731, the disclosure of which is hereinincorporated by reference.

[0038] In the subject test strips, the dry reagent composition isassociated with, e.g., present on or in, a carrier material orsubstrate. The substrate may be bibulous or non-bibulous. By bibulous ismeant a material that exhibits preferential retention of one or morecomponents as would occur, for example, in materials capable ofabsorbing or “imbibing” one or more components, as occurs inchromatographic separations. Examples of bibulous materials include, butare not limited to: nylon, untreated forms of paper, nitrocellulose andthe like which result in chromatographic separation of componentscontained in liquids, which are passed therethrough.

[0039] Alternatively, the substrate may be non-bibulous. Non-bibuloussubstrates include inert porous matrices, which provide a support forthe various members of the signal producing system, described infra, andmay have a positive charge. These matrices are generally configured toprovide a location for application of a physiological sample, e.g.,blood, and detection of the chromogenic product produced by the dye ofthe signal producing system. As such, the matrix is typically one thatis permissive of aqueous fluid flow through it and provides sufficientvoid space for the chemical reactions of the signal producing system totake place. A number of different porous matrices have been developedfor use in various analyte measurement assays, which matrices may differin terms of materials, pore sizes, dimensions and the like, whererepresentative matrices include those described in U.S. Pat. Nos.55,932,431; 5,874,099; 5,871,767; 5,869,077; 5,866,322; 5,834,001;5,800,829; 5,800,828; 5,798,113; 5,670,381; 5,663,054; 5,459,080;5,459,078; 5,441,894 and 5,212,061; the disclosures of which are hereinincorporated by reference. The dimensions and porosity of the test stripmay vary greatly, where the matrix may or may not have a porositygradient, e.g., with larger pores near or at the sample applicationregion and smaller pores at the detection region. In many embodiments,the matrix is configured as a membrane test pad and is affixed to asolid support, where the support may be a plastic (e.g., polystyrene,nylon or polyester) or metallic sheet or any other suitable materialknown in the art. Of interest in many embodiments are the test stripconfigurations disclosed in U.S. Pat. Nos. 5,972,294; 5,968,836;5,968,760; 5,902,731; 5,846,486; 5,843,692; 5,843,691; 5,789,255;5,780,304; 5,753,452; 5,753,429; 5,736,103; 5,719,034; 5,714,123;383,550; 381,591; 5,620,863; 5,605,837; 5,563,042; 5,526,120; 5,515,170;367,109; 5,453,360; 5,426,032; 5,418,142; 5,306,623; 5,304,468;5,179,005; 5,059,394; 5,049,487; 4,935,346; 4,900,666 and 4,734,360, thedisclosures of which are herein incorporated by reference.

[0040] Examples of suitable representative test strip configurations areprovided in U.S. Pat. Nos. 6,200,733 and 5,902,731, the disclosures ofwhich are herein incorporated by reference.

[0041] The subject test strips may be fabricated employing anyconvenient protocol. One convenient protocol is to contact at least thetest pad portion of the strip with an aqueous composition that includesall of the members of the reagent composition that are to be associatedwith the test pad in the final reagent test strip. Conveniently, thetest pad may be immersed in the aqueous composition, maintained thereinfor a sufficient period of time and then dried, whereby the test pad ofthe reagent test strip, which has associated therewith the reagentcomposition, is produced. As stated above, the aqueous composition willinclude the various members of the reagent composition to be associatedwith the test pad of the reagent test strip, where the various membersare present in amounts sufficient to provide for the desired amounts inthe reagent composition that is produced on the test pad. As such, wherethe electron transfer agent is non-proteinaceous, the concentration ofelectron transfer agent present in this aqueous composition typicallyranges from about 10 to 50,000, usually from about 50 to 10,000 and moreusually from about 100 to 5,000 μM. In other embodiment which containingboth non-proteinaceous and proteinaceous eletron transfer agents, theconcentration of the proteinaceous electron transfer agent present inthe aqueous composition typically ranges from about 10 to 10,000,usually from about 50 to 5,000 and more usually from about 100 to 3,000U/ml. The concentration of tetrazolium dye, e.g., tetrazolium salt,present in the aqueous composition ranges from about 3 mM to 36 mM,usually from about 6 mM to 24 mM. When present, the enzyme cofactorranges in concentration from about 1.5 mM to 28 mM, usually from about3.5 mM to 14 mM. Similarly, the analyte oxidizing agent enzyme ranges inconcentration from about 100 U to 5000 U, and usually from about 200 Uto 4000 U/ml when present. The amount of Group IIIA stabilizing agent,e.g., borax (or boric aid), when present, typically ranges from about0.1 M to about 1 M, usually from about 0.2 M to about 0.6 M. The amountof flavin stabilizing agent, e.g., FAD, FMN, when present, typicallyranges from about 1 mM to about 25 mM, usually from about 2 mM to about15 mM. See the experimental section, infra, for a more detaileddescription of a representative method for preparing the subject reagenttest strips.

[0042] Methods of Analyte Measurement

[0043] The above described signal producing systems, reagentcompositions and test strips find use in methods of detecting thepresence of, and often the amount of, i.e., the concentration of, ananalyte in a sample. A variety of different analytes may be detectedusing the subject methods, where representative analytes include thosedescribed above, e.g., alcohol, formaldehyde, glucose, glutamic acid,glycerol, beta-hydroxybutyrate, L-lactate, leucine, malic acid, pyruvicacid, steroids, etc. While in principle, the subject methods may be usedto determine the presence, and often concentration, of an analyte in avariety of different physiological samples, such as urine, tears,saliva, and the like, they are particularly suited for use indetermining the concentration of an analyte in blood or blood fractions,e.g., blood derived samples, and more particularly in whole blood, ISF(interstitial fluid).

[0044] In the subject methods, the sample and the signal producingsystem are combined into a reaction mixture, the reaction is allowed toproceed for a sufficient period to time to generate a signal indicativeof the presence of (and often amount of) analyte in the sample, and theresultant signal is detected and related to the presence of (and oftenamount of) analyte in the sample. The above steps may take place in asuitable volume containment means, e.g., cuvette, where the reagentcomposition is a fluid composition. In many embodiments, the above stepstake place on a reagent test strip as described supra.

[0045] In certain embodiments, a feature of the subject methods is thatthe detectable signal is made up of a non-washable spot that forms onthe surface of the substrate of the strip. The non-washable spot is madeup of water soluble formazan product which is tightly bound to thesubstrate surface such that it cannot be readily removed from thesurface under standard washing conditions. By standard washingconditions is meant the conditions experienced by substrate surface inanalyte detection assays where unbound component has to be removed fromthe surface. An example of standard washing conditions are thoseemployed by those of skill in the art in array based nucleic acidhybridization assays, where non-hybridized nucleic acids are removedfrom the surface of an array following a hybridization step. Suchconditions are well known to those of skill in the art. As such, afeature of the subject methods is the production of a non-washable spoton the surface of the positively charged substrate, where thenon-washable spot is made up of the water soluble formazan product.

[0046] In practicing many embodiments of the subject methods, the firststep is to apply a quantity of the physiological sample to the teststrip, where the test strip is described supra. The amount ofphysiological sample, e.g., blood that is applied to the test strip mayvary, but generally ranges from about 2 μL to 40 μL, usually from about5 μL to 20 μL. Because of the nature of the subject test strip, theblood sample size that is applied to the test strip may be relativelysmall, ranging in size from about 21 μL to 40 μL, usually from about 5μL to 20 μL. Where blood is the physiological sample, blood samples of avariety of different hematocrits may be assayed with the subjectmethods, where the hematocrit may range from about 20% to 65%, usuallyfrom about 25% to 60%.

[0047] Following application of the sample to the test strip, the sampleis allowed to react with the members of the signal producing system toproduce a detectable product, i.e., the non-washable spot, that ispresent in an amount proportional to the initial amount of the analyteof interest present in the sample. The amount of detectable product,i.e., signal produced by the signal producing system in the form of thenon-washable spot, is then determined and related to the amount ofanalyte in the initial sample. In certain embodiments, automatedinstruments that perform the above mentioned detection and relationsteps are employed. The above described reaction, detection and relatingsteps, as well as instruments for performing the same, are furtherdescribed in U.S. Pat. Nos. 4,734,360; 4,900,666; 4,935,346; 5,059,394;5,304,468; 5,306,623; 5,418,142; 5,426,032; 5,515,170; 5,526,120;5,563,042; 5,620,863; 5,753,429; 5,573,452; 5,780,304; 5,789,255;5,843,691; 5,846,486; 5,902,731; 5,968,836 and 5,972,294; thedisclosures of which are herein incorporated by reference. In therelation step, the derived analyte concentration takes into account theconstant contribution of competing reactions to the observed signal,e.g., by calibrating the instrument accordingly. KITS

[0048] Also provided by the subject invention are kits for use inpracticing the subject methods. The kits of the subject invention atleast include a signal producing system as described above, where thesignal producing system components may be combined into a single reagentcomposition or separated, e.g., present in separate containers. Incertain embodiments, the signal producing system will be present in thekits in the form of a reagent test strip, as described supra. Thesubject kits may further include a means for obtaining a physiologicalsample. For example, where the physiological sample is blood, thesubject kits may further include a means for obtaining a blood sample,such as a lance for sticking a finger, a lance actuation means, and thelike. In addition, the subject kits may include a control solution orstandard, e.g. an analyte control solution that contains a standardizedconcentration of analyte. In certain embodiments, the kits also includean automated instrument, as described above, for detecting the amount ofproduct produced on the strip following sample application and relatingthe detected product to the amount of analyte in the sample.

[0049] In addition to above mentioned components, the subject kitstypically further include instructions for using the components of thekit to practice the subject methods with the subject devices. Theinstructions for practicing the subject methods are generally recordedon a suitable recording medium. For example, the instructions may beprinted on a substrate, such as paper or plastic, etc. As such, theinstructions may be present in the kits as a package insert, in thelabeling of the container of the kit or components thereof (i.e.,associated with the packaging or subpackaging) etc. In otherembodiments, the instructions are present as an electronic storage datafile present on a suitable computer readable storage medium, e.g.CD-ROM, diskette, etc. In yet other embodiments, the actual instructionsare not present in the kit, but means for obtaining the instructionsfrom a remote source, e.g. via the internet, are provided. An example ofthis embodiment is a kit that includes a web address where theinstructions can be viewed and/or from which the instructions can bedownloaded. As with the instructions, this means for obtaining theinstructions is recorded on a suitable substrate.

[0050] The following examples are offered by way of illustration and notby way of limitation.

EXPERIMENTAL EXAMPLE 1

[0051] A 0.8 μm nylon membrane obtained from Pall Corporation (EastHills, N.Y.) was dipped into the reagent of Table 1, until saturated.The excess reagent was scraped off gently with a glass rod. Theresulting membrane was hung to dry in a 56° C. oven for 10 minutes.(Porex (0.6 mm thick) was soaked in the nitrite solution of Table 2 andthen hung to dry in a 100° C. oven for ten hours. Finally, the membranewas laminated between a polyester stock (0.4 mm Melenex® polyester fromICI America, Wilmington, Del.) and the nitrite-impregnated Porex.)

[0052] Table 2 and Porex are not Needed if Table 1 Contains NaNO₂. TABLE1 Reagent for a Glucose Test Pad Components Quantity Water 100 ml(2-[-Morpholino]ethanesulfonic acid) sodium salt MES (MW 217.2, 0.8-2.2gm Sigma, St. Louis, MO, USA) Adjust pH to 5-7 by adding 6 M HCl) Borax(MW 381.4, Sigma, St. Louis, Mo, USA 2-4 gm Gantrez 6% (Gantrez AN-139(Poly Methylvinylether-alt-Maleic 2-4 gm Anhydride, MW 1080000, Cat #41632-0, Aldrich Chemicals, Milwaukee, WI USA) Make 6% Gantrez in water,heat to 95° C. for less than 45 min. to get Gantrez 6% which is readyfor use). Adjust pH to 5.5-7 by adding 50% NaOH Triton X-305 (BASFCorporation, Mount Olive, NJ, USA) 0.5 to 2 gm. Mannitol (MW 182, Sigma,St. Louis, MO, USA) 1-10 gm Adjust pH to 5.5-7 by adding 50% NaOH SodiumNitrite (MW 69, Aldrich Chemicals, Milwaukee, WI USA) 1-5 gm.* PhenazineEthosulfate (PES, MW 334.4, Sigma, St. Louis, MO, USA 100-1000 mg WST-5(MW 1331.37, Dojindo Laboratory, Japan) 0.8-4 gm Glucose Oxidase (GO,TOYOBO) 100-1000 KU Flavin Adenine Dinucleotide (FAD) 0.2-1 gm

[0053] * If NaNO₂ is in Table 1, Table 2 is not needed. If NaNO₂ is notin Table 1, Table 2 is needed. TABLE 2 Nitrite Reagent ComponentsQuantity 10 mM Phosphate Buffer Saline, pH 7.4, (P-3813, Sigma,  70 mlSt. Louis, MO, USA) Ethanol  30 ml Sodium Nitrite (MW69, AldrichChemicals, Milwaukee,  5 gm WI, USA) Polyvinylpyrrodine (MW 40,000,Sigma, St. Louis, 200 mg MO, USA)

[0054]FIG. 1. With and without Borax, after stressed at 56° C. for 1week, tested with blood of 60%HCT and 370 mg/dL Glucose.

[0055]FIG. 2. With and without FAD, after stressed at 56° C. for 1 week,tested with blood of 60%HCT and 370 mg/dL Glucose.

[0056]FIG. 3 with and without Borax and FAD, after stressed at 56° C.for 1 week, tested with blood of 60%HCT and 370 mg/dL Glucose.

[0057] It is evident from the above results and discussion that thesubject invention provides for improvement over previous tetrazoliumdye-phenazine reagent compositions, in that it provides for a convenientway to stabilize the dye-electron transfer agent component of the systemso that light and/or heat exposure does not adversely effect the dye. Assuch, the subject invention represents a significant contribution to theart.

[0058] All publications and patents cited in this specification areherein incorporated by reference as if each individual publication orpatent were specifically and individually indicated to be incorporatedby reference. The citation of any publication is for its disclosureprior to the filing date and should not be construed as an admissionthat the present invention is not entitled to antedate such publicationby virtue of prior invention.

[0059] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

What is claimed is:
 1. A reagent composition comprising: A tetrazoliumdye; A phenazine electron transfer agent; and An effective amount of aGroup IIIA compound and/or a flavin stabilizing agent.
 2. Thecomposition according to claim 1, wherein said flavin stabilizing agentis FAD.
 3. The composition according to claim 1, wherein said Group IIIAstabilizing agent is a borate or boric acid.
 4. The compositionaccording to claim 1, wherein said reagent composition comprises ananalyte oxidizing signal producing system.
 5. The composition accordingto claim 4, wherein said analyte oxidizing signal producing systemcomprises an analyte oxidase.
 6. The composition according to claim 4,wherein said analyte oxidizing signal producing system comprises ananalyte dehydrogenase.
 7. The composition according to claim 4, whereinsaid phenazine compound is PES.
 8. The composition according to claim 4,wherein said analyte oxidizing signal producing system further comprisesan enzyme cofactor.
 9. The composition according to claim 1, whereinsaid composition is a fluid composition.
 10. The composition accordingto claim 1, wherein said composition is a dry composition.
 11. A reagenttest strip comprising: A substrate; and An analyte oxidizing signalproducing system present on said substrate, wherein said analyteoxidizing signal producing system includes: (a) a water solubletetrazolium salt; (b) a phenazine electron transfer agent; and (c) aneffective amount of Group IIIA compound and/or flavin stabilizing agent.12. The test strip according to claim 11, wherein said wherein saidflavin stablizing agent is FAD.
 13. The test strip according to claim11, wherein said Group IIIA stabilizing agent is a borate or boric acid.14. The test strip according to claim 11, wherein said analyte oxidizingsignal producing system comprises an analyte oxidase.
 15. The test stripaccording to claim 14, wherein said phenazine is PES.
 16. The test stripaccording to claim 14, wherein said analyte oxidizing signal-producingsystem further comprises an enzyme cofactor.
 17. The test stripaccording to claim 10, wherein said analyte oxidizing signal producingsystem is a glucose oxidizing signal producing system.
 18. An analytedetection or measurement system comprising: (A) A reagent test stripcomprising: (i) A substrate; and (ii) An analyte oxidizing signalproducing system present on said substrate, wherein said analyteoxidizing signal producing system includes: (a) a water solubletetrazolium salt; (b) a phenazine electron transfer agent; and (c) aneffective amount of Group IIIA compound and/or flavin stabilizing agent;and (B) An automated instrument.
 19. A method for detecting the presenceor determining the concentration of an analyte in a sample, said methodcomprising: (A) Applying said physiological sample to a reagent teststrip comprising: (i) A substrate; and (ii) An analyte oxidizing signalproducing system present on said substrate, wherein said analyteoxidizing signal producing system includes: (a) a water solubletetrazolium salt; (b) a phenazine electron transfer agent; and (c) aneffective amount of Group IIIA compound and/or flavin stabilizing agent;(B) Detecting said spot; and (C) Relating said detected spot to thepresence or concentration of said analyte in said physiological sample.20. The method according to claim 19, wherein said signal producingsystem further comprises an analyte oxidase.
 21. The method according toclaim 20, wherein said phenazine is PES.
 22. The method according toclaim 19, wherein said sample is whole blood or a derivative thereof.23. The method according to claim 19, wherein said detecting andrelating steps are carried out by an automated instrument.
 24. A kit foruse in determining the concentration of an analyte in a physiologicalsample, said kit comprising: (A) A reagent test strip comprising: (i) Asubstrate; and (ii) An analyte oxidizing signal producing system presenton said substrate, wherein said analyte oxidizing signal producingsystem includes: (a) a water soluble tetrazolium salt; (b) a phenazineelectron transfer agent; and (c) an effective amount of Group IIIAcompound and/or flavin stabilizing agent; and (B) At least one of: (i) Ameans for obtaining said physiological sample and (ii) An analytestandard.
 25. The kit according to claim 24, wherein said means forobtaining said physiological sample is a lance.
 26. The kit according toclaim 24, wherein said analyte standard comprises a standardizedconcentration of a known reagent.
 27. The kit according to claim 24,wherein said kit comprises a means for obtaining said physiologicalsample and an analyte standard.
 28. A method for stabilizing atetrazolium dye-phenazine reagent composition, said method comprising:Including in said reagent composition an effective amount of a GroupIIIA compound and/or a flavin stabilizing agent.
 29. The methodaccording to claim 28, wherein said Group IIIA compound is a borate orboric acid.
 30. The method according to claim 28, wherein said flavin isFAD.
 31. The method according to claim 28, wherein said reagentcomposition is a dry composition.
 32. The method according to claim 28,wherein said reagent composition is a wet composition.